A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis

The nucleoside analogue 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole inhibits labelling of chromosomal, high molecular weight RNA in the salivary gland cells of Chironomus tentans but does not interfere with the synthesis of ribosomal RNA and chromosomal low molecular weight RNA. When DRB2 was added after an initial labelling period (pulse-chase experiment) the radioactivity diminished preferentially in the lower molecular weight region of the HnRNA spectrum. After short chase periods the activity decreased moderately, or even increased, in the higher molecular weight region of the spectrum (75–100 S). After prolonged chases there was an overall and similar reduction in the activity in the whole HnRNA distribution. If the glands were preincubated in DRB for a short period before exposure to radioactive precursors, the label was again diminished more in HnRNA of low molecular weight than in that of higher molecular weight. When α-amanitin or actinomycin D, both known to be inhibitors of RNA chain elongation, replaced DRB in pulse-chase experiments, labelling of HnRNA was depressed in all size classes to the same extent. The accumulated data suggest that DRB acts, in explanted salivary gland cells, at the polymerase level by interfering with the initiation of chromosomal HnRNA synthesis.     

Change in the concentration and intensity of synthesis of RNA in cell cultures of Chironomus thummi during metamorphosis

The content and intensity of incorporation of 3H-uridine in RNA of chromosomes, nucleoli and cytoplasm isolated by microsurgery from the salivary glands of larvae and prepupae of Chironomus thummi were studied following the incubation of salivary glands in the Cannon's medium with 3H-uridine. It was shown that during metamorphosis the content of RNA and intensity of 3H-uridine incorporation decrease in the nucleolus and cytoplasm in a prepupa, as compared with a larva, and suffer no changes in chromosomes in spite of much larger size of many puffs in a prepupa. The patterns of RNA synthesis in the salivary glands of larvae during metamorphosis are discussed.     

Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs

The salivary glands and other tissues from Drosophila melanogaster were dissected at various times throughout the prepupal period, as well as after heat shocks and ecdysterone treatments, and the proteins labelled by incubating the isolated tissues with [³⁵S]methionine were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel. The labelled band patterns from salivary gland, as seen on the autoradiograph of the gel, showed striking variations, in a manner remarkably similar to variations in puff patterns during the same prepupal period. In proteins from Malpighian tubes, the pattern of bands varied to a lesser extent and in brain only a few components were modified.Heat shock brought about the appearance of a number of new bands, while others were reduced in intensity. This effect was observed with all the tissues examined, salivary glands, brain and Malpighian tubes, as well as wing imaginal discs, tissue lacking polytene chromosomes. The six most heavily labelled bands induced by heat shock represent about 30%, and one component alone represents over 15%, of the total label in the sample, as seen in salivary glands, brain and Malpighian tubes. The synthesis of RNA at puff sites was investigated after heat shock by [³H]uridine labelling. By correlating the amount of [³H]uridine in some puffs with the level of [³⁵S]methionine in some bands a tentative relation is suggested in a few instances.The effect of ecdysterone treatment was also studied in the salivary glands. Changes in a number of protein bands were noticed, though they were much less pronounced than those following heat shock.     

Localisation of epidermal growth factor receptor in mucous cells of human salivary glands

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.     

MASS PREPARATION OF NUCLEI FROM THE LARVAL SALIVARY GLANDS OF DROSOPHILA HYDEI

A method has been developed for isolating gram quantities of salivary glands from late third instar larvae of Drosophila hydei. The isolated glands have a normal appearance and incorporate RNA and DNA precursors normally. Nuclei can be isolated from these glands in 90% yield with the use of detergents. These nuclei contain morphologically normal giant polytene chromosomes.     

Noncoordinate expression of Drosophila glue genes: Sgs-4 is expressed at many stages and in two different tissues.

The glue genes of Drosophila melanogaster comprise a family of genes expressed at high levels in the salivary glands of late third instar larvae in response to the insect hormone ecdysone. We present evidence that, in contrast to the other glue genes, Sgs-4 is turned on throughout Drosophila development and is not expressed exclusively in the larval salivary glands. Larvae transformed with an Sgs-4/Adh (alcohol dehydrogenase) hybrid gene exhibit Sgs-4-directed Adh expression in the larval proventriculus as well as in the salivary glands as early as the first instar. Sgs-4-specific RNA can be detected at very low levels during all stages of development. During late third instar, levels of Sgs-4 RNA in the salivary glands increase several-thousand-fold, thereby accounting for the large amounts of Sgs-4 protein present in the glue produced by the salivary glands. This pattern of expression is unique to the Sgs-4 gene. While expression of several of the other glue genes can be detected in embryos and early larvae, they appear to be expressed neither throughout development nor in the larval proventriculus. Appearance of the glue gene RNAs in mid third instar salivary glands is noncoordinate, even for the chromosomally clustered genes Sgs-3, Sgs-7, and Sgs-8.     

The effect of perchloric acid fixation on DNA preservation in the salivary gland of Bradysia hygida

In Bradysia hygida (Diptera, Sciaridae) well spread polytene chromosomes, free of cytoplasm, and with good morphology are consistently obtained if before squashing in acetic acid solution, the salivary glands are fixed in 7% perchloric acid containing small amounts of ferric ions (SAUAIA et al. 1971). We show here that, with regard to the preservation of total incorporated 3H-thymidine and with regard to the relative autoradiographic labelling of expanded chromosome regions as compared to the labelling of a non-expanded one, this method is equivalent to fixing the salivary glands in ethanol-acetic (3:1). We show also that if this kind of preparation is subject to mild acid hydrolysis a small amount of the total 3H-labelled material may be lost.     

Puff induction in permeabilized Drosophila salivary glands in a chemically defined medium

We showed previously that digitonin-permeabilized salivary glands form prominent puffs in response to ecdysterone only when the incubation medium is supplemented with a homogenate of intact glands. To develop a chemically defined medium that supports puff formation in permeabilized salivary glands, we examined the requirement of ribonucleoside triphosphates (NTPs), precursors of RNA synthesis, for puff formation in permeabilized salivary glands. We found that prominent ecdysone puffs were induced in permeabilized salivary glands when the concentration of each NTP in the medium was higher than 0.5 mM. The puff size was significantly reduced if the volume of the medium were more than 2.0 microliter per gland. This suggests the existence of a factor(s), in addition to NTPs, which is required for puff formation and is diffusible from permeabilized glands.     

Comparison of differentially expressed genes in the salivary glands of male ticks,Amblyomma americanum and Dermacentor andersoni

Genes expressed differentially in the salivary glands of unfed and fed male ticks, Amblyomma americanum (L.), were identified, cloned and sequenced, and some were compared with those expressed in the salivary glands of Dermacentor andersoni. Total protein and RNA increased sixfold in the salivary glands of fed male A. americanum, while in fed male D. andersoni salivary glands, RNA increased approximately 3.5 times. Feeding D. andersoni in the presence of females increased total RNA by 25% over those fed in the absence of females. Complementary DNAs were synthesized from RNA obtained from unfed and fed ticks and amplified using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) with three different primers in separate reactions. Differential display showed clear banding differences between the fed and the unfed ticks in A. americanum and D. andersoni. Sixty-one cDNA fragments that appeared to be from differentially expressed genes in A. americanum were isolated, cloned and sequenced. Hybridization reactions with labeled cDNA probes confirmed the differential expression of many of the genes in unfed and fed ticks' salivary glands; however, many of the bands contained more than one fragment and some of the fragments isolated from apparently differential bands were not specific. Sequences for 28 of the cDNA fragments (150-600 nucleotides in length) demonstrated similarity to genes in the databases, but nine of these were similar to sequences of unknown function. Some of the gene fragments identified may be important to tick feeding or tick salivary gland physiology, including a histamine-binding protein, an organic ion transporter, an apoptosis inhibitor, a cathepsin-B-like cysteine protease, proteins involved in gene regulation and several proteins involved in protein synthesis. Cross-hybridization of identified cDNAs from A. americanum with cDNA probes synthesized from D. andersoni total RNA did not show significant similarity between the two species.     

The Short-Term Effects of A Single Injection of Isoproterenol On Proliferation In the Submandibular Gland, Parotid Gland and Oesophagus In Vivo

Abstract. Mitotic and labelling indices were studied in the submandibular, parotid and oesophageal cells of male mice within the first 6 hr (but particularly within the 1st hr) of a single injection of isoproterenol or saline, using the metaphase arrest agent (vincristine) which was previously tested for efficacy in submandibular gland. There was a significant increase in the metaphase index of the salivary glands over control values 5, 15, 30, 45 and 60 min after isoproterenol. In contrast, there were no significant changes in the metaphase index of basal cells of the oesophagus. There was no significant change in the labelling index in isoproterenol-treated mice in comparison with saline-injected control animals. Possible explanations for the rapid mitotic response in murine salivary glands are considered; a rapid efflux from G₂ into mitosis is thought to be the most likely.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Replication in Drosophila chromosomes

The temporal order of replication of specific sites in polytene chromosomes from salivary glands and gastric caeca of Drosophila nasuta larvae was compared using ³H-thymidine autoradiography. Labelling of different cytological regions in segments of chromosome 2R (section 47 A to 49 C) and chromosome 3 (section 80 A to 82 C) was examined in detail in nuclei showing late S-period labelling (2 D and 1D types) in both cell types. The different labelling sites (22 on the 2R segment and 38 on the chromosome 3 segment) are cytologically similar in the two cell types. However, there are profound differences in the labelling frequencies of certain sites in polytene nuclei from salivary glands and gastric caeca during the late S-phase. This suggests that even though a comparable number of chromosomal replicating units operates in the two polytene cell types, the temporal order of completion of replication differs.     

The synthesis and origin of chloroplast low-molecular-weight ribosomal ribonucleic acid in spinach.

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA species which co-electrophorese with 5-S rRNA and tRNA, but show very little incorporation into 4.5-S rRNA. Chloroplast 4.5-S rRNA is labelled in vivo after a distinct lag period relative to 5-S rRNA and tRNA. The kinetics of labelling in vivo of chloroplast 5-S rRNA are similar to those of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, whereas the kinetics of labelling of the 4.5-S rRNAare similar to those of mature 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs. Chloramphenicol inhibits the labelling of chloroplast 4.5-S rRNA in vivo, and concomitantly inhibits the processing of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, but has little effect on the appearance of label in chloroplast 5-S rRNA. DNA/RNA hybridization using 125I-labelled RNAs suggests that chloroplast DNA contains a 2--3-fold excess of 4.5-S and 5-S rRNA genes relative to the high-molecular-weight rRNA genes. Competition hybridization experiments show that the immediate precursor to the 1.05 x 10(6)-Mr rRNA effectively competes with 125I-labelled 4.5-S rRNA for hybridization with chloroplast DNA, and is therefore a likely candidate for a common precursor to both the 1.05 x 10(6)-Mr and 4.5-S rRNAs.     

Quantitative histoenzymologic characteristics of the submaxillary salivary glands of white rats during an ovarian cycle]

Ovarian cycle in albino rats was applied to ascertain the problem of the relationship between the salivary and endocrine glands, and also of the extent of participation of individual components of the salivary glands with different functional orientation in the endocrine regulation of individual components of the salivary glands. The content of protein, mucopolysaccharides, DNA, and RNA, the activity of NAD- and NADP-diaphorase, alkaline phosphatase, malate and isocitrate dehydrogenase, alpha-leucine-aminopeptidase was studied. Cytospectrophotometric analysis showed that synchronous changes in the activity of the enzymes under study occurred in all the portions of the salivary glands, depending on the ovarian cycle phases. Of the four successive phases of the cycle the greatest activity of the enzymes and of the protein and mucopolysaccharide content was noted during the proestrus and metaestrus. Different metabolic processes were observed in the salivary ducts in comparison with other parts of the gland; this was apparently connected with peculiarities of the secretion and hormone production.